Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.

Formation of specialized reaction spaces prevents interference between distinct cellular pathways. Peroxisomes are cellular compartments involved in a large diversity of metabolic processes. How peroxisomes differentiate into subpopulations and by which mechanism intraorganellar domains are formed remains largely elusive. Here, we report on enzymes from the fungus Ustilago maydis, which accumulate inside of peroxisomal subdomains. We describe a short peptide motif (Tyr-Ile-Ile-Val) sufficient to trigger focal localization. Mining for proteins with similar motifs uncovered several peroxisomal matrix proteins that accumulate in intraorganellar foci. These foci are enriched in the enzyme urate oxidase – a typical constituent of the paracrystalline core of peroxisomes. Upon peroxisome proliferation uneven distribution of focal structures results in the formation of peroxisome subpopulations with different protein content. The underlying principle of subdomain formation is evolutionary conserved in mammalian peroxisomes and formation of similar foci was also observed inside of mitochondria. We propose that peroxisomal proteins show an individual propensity to self-assemble. This formation of protein aggregates appears to be a ubiquitous driving force to spatially organize the peroxisomal proteome.